Biosynthesis of new alpha-bisabolol derivatives through a synthetic biology approach

La biologie de synthèse permet désormais la production de nouvelles molécules n’existant pas dans la nature. Au cours de cette thèse, nous nous sommes focalisés sur la diversification de produits issus du (+)-epi-α-bisabolol. Cette molécule issue de la plante Lippia dulcis appartient à la vaste famille des sesquiterpènes qui recèle de nombreuses activités biologiques. En particulier, le (+)-epi-α-bisabolol est le précurseur de l’hernandulcine, un édulcorant intense. Cependant, la dernière étape d’oxydation conduisant à ce composé demeure inconnue.Plutôt que de rechercher la (ou les enzymes) intervenant dans la voie de biosynthèse de l’hernandulcine chez L. dulcis, nous avons choisi de mettre à profit la promiscuité connue de cytochromes P450 pour diversifier les molécules issues du (+)-epi-α-bisabolol ou de reproduire la voie de synthèse naturelle. Tout d’abord, une souche châssis adaptée au criblage in vivo a été construite en exprimant la (+)-epi-α-bisabolol synthase (BBS) et une NADPH cytochrome P450 réductase. Puis, le criblage in vivo chez la levure a montré que des cytochromes P450 (CYPs) parmi notre banque de 25 enzymes impliquées dans le métabolisme des xénobiotiques (CYPs animaux) oxydaient le (+)-epi-α-bisabolol. Parmi ces produits, le 14-hydroxy-(+)-epi-α-bisabolol a été purifié et caractérisé par RMN tandis que la structure probable d’un second produit a été obtenue (9-hydroxy-(+)-epi-α-bisabolol). En parallèle, la production de produits hydroxylés (+)-epi-α-bisabolol a été optimisée notamment par l’addition d’une copie génomique supplémentaire de BBS, avec un titre de de produit hydroxylé de 64 mg/L. Ainsi, nous avons démontré le potentiel des CYPs du métabolisme des xénobiotiques dans la synthèse de nouvelles molécules issues des sesquiterpènes.Parmi les enzymes identifiées, deux enzymes homologues, CYP2B6 et CYP2B11 ont catalysé la formation de produits différents à partir du (-)-α-bisabolol et du trans,trans-farnésol. Pour identifier les possibles déterminants moléculaires responsables de la spécificité de chacune de ces deux enzymes, des chimères échangeant divers éléments de structure secondaire de CYP2B6 dans CYP2B11 ont été comparées. Ainsi, la spécificité ne peut pas être expliquée par un seul élément de la structure secondaire mais plus probablement par des déterminants disséminés en divers endroits des séquences protéiques des deux enzymes.Afin d’étendre la diversité de molécules produites à partir de l’α-bisabolol et de l’hernandulcine et de produire des composés aux propriétés physico-chimiques améliorées (solubilité, pouvoir sucrant…) des essais de glycosylation par une glucosyltransférase de plante (UGT93B16) et par des glucuronosyltransférases humaines ont été menés. UGT93B16 a démontré son potentiel en glucosylant l’α-bisabolol et l’hernandulcine. La bioconversion de ces molécules en utilisant E. coli and S. cerevisiae a également montré une meilleure conversion chez la levure. Concernant les UDP-glucuronosyltransférases, des essais enzymatiques ont mis en évidence une activité d’UGT1A9, d’UGT2B4 et d’UGT2B7 pour l’α-bisabolol et l’hernandulcine. Toutefois, l’introduction de ces enzymes dans la levure n’a pas été permis la production de sesquiterpènes glucuronylés à un niveau détectable. Pour résumer, nous avons prouvé la faisabilité de la production de nouveaux sesquiterpènes glycosylés soit par voie enzymatique soit par bioconversion chez deux microorganismes couramment utilisés en biotechnologie.En conclusion, les approches utilisées au cours de cette thèse ont montré l’intérêt du criblage d’enzymes promiscuitaires pour l’α-bisabolol et la production de nouvelles molécules. Nous avons exploré les limites de notre souche châssis et une régulation plus fine du métabolisme de S. cerevisiae pourrait améliorer les titres obtenus. Enfin, le couplage des étapes impliquant un cytochrome P450 et une glucosyltransférase est désormais envisageable afin de créer une voie encore plus orthogonale.The rise of synthetic biology now enables the production of new to nature molecules. In the frame of this thesis we focused on the diversification of the (+)-epi-α-bisabolol scaffold. This molecule coming from the plant Lippia dulcis belongs to the vast family of sesquiterpenes. While sesquiterpenes possess diverse biological activities, (+)-epi-α-bisabolol is the precursor of hernandulcin, an intense sweetener. However, the last oxidative step(s) of the hernandulcin biosynthetic pathway remain elusive.Rather than seeking the native oxidase responsible for hernandulcin synthesis among L. dulcis enzymes we selected oxidative enzymes known to be promiscuous and that could functionalize (+)-epi-α-bisabolol in order to i) generate diversity from (+)-epi-α-bisabolol; ii) hopefully identify an oxidative enzyme catalyzing hernandulcin synthesis. First, a yeast chassis strain enabling the in vivo screening of cytochromes P450 (CYPs) was constructed by coexpressing two key enzymes: the (+)-epi-α-bisabolol synthase (BBS) and the NADPH cytochrome P450 reductase. Then, the in vivo screening assays revealed that 5 CYPs out of our library of 25 animal CYPs involved in xenobiotic metabolism oxidized (+)-epi-α-bisabolol and produced new hydroxylated regioisomers. Of the oxidized products, the structure of one compound, 14-hydroxy-(+)-epi-α-bisabolol, was fully elucidated by NMR while the probable structure of a second one was determined (9-hydroxy-(+)-epi-α-bisabolol). In parallel, the production of (+)-epi-α-bisabolol derivatives was enhanced through addition of a supplementary genomic copy of BBS that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism CYPs can be used to produce novel compounds from a sesquiterpene scaffold.Furthermore, different products were obtained with two homologous enzymes i.e. CYP2B6 and CYP2B11. This prompted us to study the molecular determinants putatively responsible for enzyme regiospecificity. From chimeric enzymes composed of secondary structure elements originating from CYP2B6 and CYP2B11 we were not able to identify specific motifs that could explain the CYPs regiospecificity. This approach suggests that the molecular determinants cannot be attributed to specific structural elements of the two enzymes but are rather widespread in the protein sequences.In order to generate a wider molecular diversity from α-bisabolol or hernandulcin, and synthesize molecules with different physico-chemical and biological properties (i.e. solubility, sweetening power etc.), we attempted the glycosylation of these compounds using a plant glucosyltransferase (UGT93B16) or human glucuronosyltransferases. UGT93B16 was found to glucosylate both (-)-α-bisabolol and hernandulcin. In addition, we carried out whole cell catalysis using E. coli and S. cerevisiae as recombinant producers of UGT93B16. Comparison of the two microbial hosts showed that glycosylation using yeast cells was more efficient. In parallel, we investigated α-bisabolol glucosylation by human UDP-glucuronosyltransferases involved in the xenobiotic metabolism. In vitro enzymatic assays demonstrated a weak activity of UGT1A9, UGT2B4 and UGT2B7 towards α-bisabolol and hernandulcin. However, their introduction in yeast failed to produce detectable amounts of glucuronide products. In summary, we proved the feasibility of producing new to nature sesquiterpene glucosides using either enzyme-based assay or bioconversion in two different hosts that are widely used in biotechnology.To conclude, the approaches used in this thesis highlight the assets of screening promiscuous enzymes for the production of new molecules from α-bisabolol. We also explored the limits of our chassis strain and a tighter regulation of S. cerevisiae metabolism could improve the production (+)-epi-α-bisabolol oxidized products. Finally, the coupling in yeast of cytochrome P450 and glucosyltransferase steps can now be envisioned

Biosynthèse de nouveaux dérivés de l'alpha-bisabolol par une approche de biologie synthèse

The rise of synthetic biology now enables the production of new to nature molecules. In the frame of this thesis we focused on the diversification of the (+)-epi-α-bisabolol scaffold. This molecule coming from the plant Lippia dulcis belongs to the vast family of sesquiterpenes. While sesquiterpenes possess diverse biological activities, (+)-epi-α-bisabolol is the precursor of hernandulcin, an intense sweetener. However, the last oxidative step(s) of the hernandulcin biosynthetic pathway remain elusive.Rather than seeking the native oxidase responsible for hernandulcin synthesis among L. dulcis enzymes we selected oxidative enzymes known to be promiscuous and that could functionalize (+)-epi-α-bisabolol in order to i) generate diversity from (+)-epi-α-bisabolol; ii) hopefully identify an oxidative enzyme catalyzing hernandulcin synthesis. First, a yeast chassis strain enabling the in vivo screening of cytochromes P450 (CYPs) was constructed by coexpressing two key enzymes: the (+)-epi-α-bisabolol synthase (BBS) and the NADPH cytochrome P450 reductase. Then, the in vivo screening assays revealed that 5 CYPs out of our library of 25 animal CYPs involved in xenobiotic metabolism oxidized (+)-epi-α-bisabolol and produced new hydroxylated regioisomers. Of the oxidized products, the structure of one compound, 14-hydroxy-(+)-epi-α-bisabolol, was fully elucidated by NMR while the probable structure of a second one was determined (9-hydroxy-(+)-epi-α-bisabolol). In parallel, the production of (+)-epi-α-bisabolol derivatives was enhanced through addition of a supplementary genomic copy of BBS that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism CYPs can be used to produce novel compounds from a sesquiterpene scaffold.Furthermore, different products were obtained with two homologous enzymes i.e. CYP2B6 and CYP2B11. This prompted us to study the molecular determinants putatively responsible for enzyme regiospecificity. From chimeric enzymes composed of secondary structure elements originating from CYP2B6 and CYP2B11 we were not able to identify specific motifs that could explain the CYPs regiospecificity. This approach suggests that the molecular determinants cannot be attributed to specific structural elements of the two enzymes but are rather widespread in the protein sequences.In order to generate a wider molecular diversity from α-bisabolol or hernandulcin, and synthesize molecules with different physico-chemical and biological properties (i.e. solubility, sweetening power etc.), we attempted the glycosylation of these compounds using a plant glucosyltransferase (UGT93B16) or human glucuronosyltransferases. UGT93B16 was found to glucosylate both (-)-α-bisabolol and hernandulcin. In addition, we carried out whole cell catalysis using E. coli and S. cerevisiae as recombinant producers of UGT93B16. Comparison of the two microbial hosts showed that glycosylation using yeast cells was more efficient. In parallel, we investigated α-bisabolol glucosylation by human UDP-glucuronosyltransferases involved in the xenobiotic metabolism. In vitro enzymatic assays demonstrated a weak activity of UGT1A9, UGT2B4 and UGT2B7 towards α-bisabolol and hernandulcin. However, their introduction in yeast failed to produce detectable amounts of glucuronide products. In summary, we proved the feasibility of producing new to nature sesquiterpene glucosides using either enzyme-based assay or bioconversion in two different hosts that are widely used in biotechnology.To conclude, the approaches used in this thesis highlight the assets of screening promiscuous enzymes for the production of new molecules from α-bisabolol. We also explored the limits of our chassis strain and a tighter regulation of S. cerevisiae metabolism could improve the production (+)-epi-α-bisabolol oxidized products. Finally, the coupling in yeast of cytochrome P450 and glucosyltransferase steps can now be envisioned.La biologie de synthèse permet désormais la production de nouvelles molécules n’existant pas dans la nature. Au cours de cette thèse, nous nous sommes focalisés sur la diversification de produits issus du (+)-epi-α-bisabolol. Cette molécule issue de la plante Lippia dulcis appartient à la vaste famille des sesquiterpènes qui recèle de nombreuses activités biologiques. En particulier, le (+)-epi-α-bisabolol est le précurseur de l’hernandulcine, un édulcorant intense. Cependant, la dernière étape d’oxydation conduisant à ce composé demeure inconnue.Plutôt que de rechercher la (ou les enzymes) intervenant dans la voie de biosynthèse de l’hernandulcine chez L. dulcis, nous avons choisi de mettre à profit la promiscuité connue de cytochromes P450 pour diversifier les molécules issues du (+)-epi-α-bisabolol ou de reproduire la voie de synthèse naturelle. Tout d’abord, une souche châssis adaptée au criblage in vivo a été construite en exprimant la (+)-epi-α-bisabolol synthase (BBS) et une NADPH cytochrome P450 réductase. Puis, le criblage in vivo chez la levure a montré que des cytochromes P450 (CYPs) parmi notre banque de 25 enzymes impliquées dans le métabolisme des xénobiotiques (CYPs animaux) oxydaient le (+)-epi-α-bisabolol. Parmi ces produits, le 14-hydroxy-(+)-epi-α-bisabolol a été purifié et caractérisé par RMN tandis que la structure probable d’un second produit a été obtenue (9-hydroxy-(+)-epi-α-bisabolol). En parallèle, la production de produits hydroxylés (+)-epi-α-bisabolol a été optimisée notamment par l’addition d’une copie génomique supplémentaire de BBS, avec un titre de de produit hydroxylé de 64 mg/L. Ainsi, nous avons démontré le potentiel des CYPs du métabolisme des xénobiotiques dans la synthèse de nouvelles molécules issues des sesquiterpènes.Parmi les enzymes identifiées, deux enzymes homologues, CYP2B6 et CYP2B11 ont catalysé la formation de produits différents à partir du (-)-α-bisabolol et du trans,trans-farnésol. Pour identifier les possibles déterminants moléculaires responsables de la spécificité de chacune de ces deux enzymes, des chimères échangeant divers éléments de structure secondaire de CYP2B6 dans CYP2B11 ont été comparées. Ainsi, la spécificité ne peut pas être expliquée par un seul élément de la structure secondaire mais plus probablement par des déterminants disséminés en divers endroits des séquences protéiques des deux enzymes.Afin d’étendre la diversité de molécules produites à partir de l’α-bisabolol et de l’hernandulcine et de produire des composés aux propriétés physico-chimiques améliorées (solubilité, pouvoir sucrant…) des essais de glycosylation par une glucosyltransférase de plante (UGT93B16) et par des glucuronosyltransférases humaines ont été menés. UGT93B16 a démontré son potentiel en glucosylant l’α-bisabolol et l’hernandulcine. La bioconversion de ces molécules en utilisant E. coli and S. cerevisiae a également montré une meilleure conversion chez la levure. Concernant les UDP-glucuronosyltransférases, des essais enzymatiques ont mis en évidence une activité d’UGT1A9, d’UGT2B4 et d’UGT2B7 pour l’α-bisabolol et l’hernandulcine. Toutefois, l’introduction de ces enzymes dans la levure n’a pas été permis la production de sesquiterpènes glucuronylés à un niveau détectable. Pour résumer, nous avons prouvé la faisabilité de la production de nouveaux sesquiterpènes glycosylés soit par voie enzymatique soit par bioconversion chez deux microorganismes couramment utilisés en biotechnologie.En conclusion, les approches utilisées au cours de cette thèse ont montré l’intérêt du criblage d’enzymes promiscuitaires pour l’α-bisabolol et la production de nouvelles molécules. Nous avons exploré les limites de notre souche châssis et une régulation plus fine du métabolisme de S. cerevisiae pourrait améliorer les titres obtenus. Enfin, le couplage des étapes impliquant un cytochrome P450 et une glucosyltransférase est désormais envisageable afin de créer une voie encore plus orthogonale

The role of the methyltransferase domain of bifunctional restriction enzyme RM.BpuSI in cleavage activity.

Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca(2+), SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca(2+), substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca(2+) and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI

Mutation of N406 in the NPPY motif abolishes MTase and cleavage activity.

<p>(A) Fluorescence spectra of WT and N406A RM.BpuSI. The high similarity of the two spectra indicated that mutation N406A does not induce significant conformation change to RM.BpuSI. Titration of SAM or SIN into mutant N406A does not show specific changes in fluorescence (data not shown). (B) Titration of SAM or SIN into WT RM.BpuSI in the presence of 400 μM ANS. The data points are fitted to a hyperbolic function and K_d values are found to be 76.4 and 71.1 μM for SAM and SIN, respectively. (C) Two-fold dilutions of WT or N406A RM.BpuSI was incubated with 1 μg of λ DNA in the presence or absence of 160 μM SAM at 37°C for 1 h. While WT exhibits enhanced cleavage activity in the presence of SAM, mutant N406A does not exhibit cleavage activity in the presence or absence of SAM.</p

Oligonucleotide duplexes used in this study and their susceptibility to RM.BpuSI cleavage.

<p>(A) Sub01 is a 49-bp synthetic oligonucleotide duplex containing a single BpuSI site. Site-specific modified versions of sub01 were also synthesized. M1-modified sub01 (M1_mod) contains a N6mA (red) on the top strand. M2-modified sub01 (M2_mod) contains a m5C (red) on the bottom strand. Oligonucleotide duplex M1/M2_mod contains both modified bases. Pdt01 and pdt02 are the RM.BpuSI cleavage products. (B) RM.BpuSI REase activity. RM.BpuSI cleaved unmodified sub01 into expected fragments. Substrates containing M2- or M1/M2-modifications were not cleaved. A small amount of cleavage product is observed with M1-modified substrate.</p

Mutation N406A results in conformations incompatible to cleavage upon binding with Ca²⁺ and/or substrate.

<p>The fluorescence spectra of RM.BpuSI WT or mutant N406A alone and with sub01 (A) or CaCl₂ (B) or with sub01 and CaCl₂ (C) are shown. Mutant N406A adopts conformations very different from that of the WT in the presence of the ligands. </p

Effect of SAM and SIN on the steady state kinetics of cleavage activity.

<p>Steady state kinetics experiments of RM.BpuSI cleavage activity were carried out by incubating 200 nM of purified RM.BpuSI protein with 1.25 to 25 μM of FAM-labeled sub01 at 37°C for 1 h in the presence or absence of 160 μM of SAM or SIN. The initial rate (v) for each substrate concentration was plotted against substrate concentration. K_m and V_max values were obtained by fitting the data points to the Michaelis-Menten equation. Error bars indicate the standard deviation of the fitting of the initial linear rate of each data point.</p

Fluorescence spectra of RM.BpuSI-bound ANS as surrogate of protein conformations.

<p>(A) Pre-cleavage and cleavage states. Fluorescence spectra of RM.BpuSI alone, with Ca²⁺, sub01 and sub01+Ca²⁺ are shown. The Ca²⁺, sub01 and sub01+Ca²⁺ spectra have significant lower maximum emission intensity from that of the WT RM.BpuSI alone, suggesting substantial conformational changes upon binding to the ligands. The Ca²⁺, sub01 and sub01+Ca²⁺ spectra resembled each other in shape and emission maximum and are inferred as representing the pre-cleavage/cleavage state. (B) and (C) SIN induces the MTase conformation. Fluorescence spectra of RM.BpuSI alone, with sub01, SIN and SIN and sub01 are shown. In the presence of SIN, RM.BpuSI adopts a conformation different from the SIN or Ca²⁺-bound conformation. The RM.BpuSI+sub01+SIN spectrum exhibits a higher maximum emission intensity than the RM.BpuSI+sub01 spectrum (B). The fluorescence spectra of RM.BpuSI alone, with SIN, pdt01, SIN+sub01 or SIN+pdt01 are shown (C). The spectra of sub01+SIN and pdt01+SIN are indistinguishable, suggesting an MTase state conformation. The spectrum of pdt01 is also indistinguishable from the SIN-bound spectra, suggesting that binding of pdt01 in the absence of divalent metal ions induces the MTase state. (D) The RM.BpuSI+sub01+Ca²⁺+SIN spectrum is highly similar to that without SIN, especially in the lower emission wavelengths, suggesting that the presence of SIN does not induce significant conformational change to the RM.BpuSI-M²⁺-substrate complex. The fluorescence spectrum of RM.BpuSI+pdt01+Ca²⁺+SIN is significantly different from any of the spectra, suggesting that the enzyme undergoes more conformational changes after the cleavage reaction and remains bound to the product. (E) In the presence of sub01, SIN and Ca²⁺, the fluorescence spectrum of RM.BpuSI-bound ANS resembles the RM.BpuSI+sub01+SIN+Ca²⁺ (cleavage conformation) spectrum but not the RM.BpuSI+sub01+SIN spectrum (MTase conformation). (F) Sub01 was titrated into a solution containing 2.5 μM RM.BpuSI and 2 mM CaCl₂ in the presence or absence of 160 μM SIN. Fluorescence change (ΔF) relative to zero sub01 conditions was plotted against the final concentrations of sub01. The dissociation constant K_d is 0.17 or 1.9 μM in the presence or absence of SIN, respectively.</p

Synthetic Derivatives of (+)- epi -α-Bisabolol Are Formed by Mammalian Cytochromes P450 Expressed in a Yeast Reconstituted Pathway

International audienceIdentification of the enzyme(s) involved in complex biosynthetic pathways can be challenging. An alternative approach might be to deliberately diverge from the original natural enzyme source and use promiscuous enzymes from other organisms. In this paper, we have tested the ability of a series of human and animal cytochromes P450 involved in xenobiotic detoxification to generate derivatives of (+)-epi-alpha-bisabolol and attempt to produce the direct precursor of hernandulcin, a sweetener from Lippia dulcis for which the last enzymatic steps are unknown. Screening steps were implemented in vivo in S. cerevisiae optimized for the biosynthesis of oxidized derivatives of (+)-epi-alpha-bisabolol by coexpressing two key enzymes: the (+)-epi-alpha-bisabolol synthase and the NADPH cytochrome P450 reductase. Five out of 25 cytochromes P450 were capable of producing new hydroxylated regioisomers of (+)-epi-alpha-bisabolol. Of the new oxidized bisabolol products, the structure of one compound, 14-hydroxy-(+)-epi-alpha-bisabolol, was fully elucidated by NMR while the probable structure of the second product was determined. In parallel, the production of (+)-epi-alpha-bisabolol derivatives was enhanced through the addition of a supplementary genomic copy of (+)-epi-alpha-bisabolol synthase that augmented the final titer of hydroxylated product to 64 mg/L. We thus demonstrate that promiscuous drug metabolism cytochromes P450 can be used to produce novel compounds from a terpene scaffold